METHODS FOR ISOLATING AND CHARACTERIZING ENDOGENOUS mRNA-PROTEIN (mRNP) COMPLEXES

ABSTRACT

Cellular mRNA-protein (mRNP) complexes are partitioned in vivo by contacting a biological sample with at least one ligand that specifically binds at least one component of a mRNP complex. Suitable biological samples comprise at least one mRNA-protein (mRNP) complex and include cell cultures, cell extracts, and whole tissue, including tumor tissue. Ligands include antibodies that specifically bind RNA-binding or RNA-associated proteins present in the mRNP complex. The mRNP complex is separated by binding the ligand with a binding molecule specific for the ligand, where the binding molecule is attached to a solid support. The mRNP complex is collected by removing the mRNP complex from the solid support. After collecting the mRNP complex, the mRNA bound within the complex may be characterized and identified. Subsets of the total mRNA population of a cell may accordingly be characterized, and a gene expression profile of the cell obtained.

RELATED APPLICATIONS

This application is a divisional application based on a restrictionrequirement in co-pending U.S. patent application Ser. No. 10/238,306,filed on Sep. 10, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/750,401, filed on Dec. 28, 2000, now issued asU.S. Pat. No. 6,635,422, which claims the benefit of U.S. ProvisionalApplication No. 60/173,338, filed Dec. 28, 1999, the contents of whichare hereby incorporated in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under grant number R01CA79907 from the National Institutes of Health. The United Statesgovernment has certain rights to this invention.

FIELD OF THE INVENTION

This invention relates generally to post-transcriptional regulation andmethods of profiling gene expression.

BACKGROUND OF THE INVENTION

Many diseases are genetically based, and the genetic background of eachindividual can have a profound effect on his or her susceptibility todisease. The relatively new field of functional genomics has providedresearchers with the ability to determine the functions of proteinsbased upon knowledge of the genes that encode the proteins. A major goalof functional genomics is to identify gene products that are suitabletargets for drug discovery. Such knowledge can lead to a basis fortarget validation if it is demonstrated that the target of interest hasan essential function in a disease. Accordingly, a need exists todevelop methods that allow profiling of the gene expression state ofcells and tissues in order to understand the consequences of genetics ongrowth and development.

Understanding global gene expression at the level of the whole cellrequires detailed knowledge of the contributions of transcription,pre-mRNA processing, mRNA turnover and translation. Although the sumtotal of these regulatory processes in each cell accounts for its uniqueexpression profile, few methods are available to independently assesseach process en masse.

The expression state of genes in a complex tissue or tumor is generallydetermined by extracting messenger RNAs from samples (e.g., wholetissues) and analyzing the expressed genes using cDNA libraries,microarrays or serial analysis of gene expression (SAGE) methodologies.See, e.g., Duggan, et al., (1999) Nature Genetics 21, 10-14.; Gerhold,et al., (1999) Trends in Biochemical Sciences 24, 168-173; Brown, etal., (1999) Nature Genetics 21, 38-41; Velculescu, et al., (1995)Science 270, 484-487 Velculescu, et al. (1997) Cell 88, 243-251. Inorder to determine the gene expression profile of any single cell typewithin a tissue or tumor or to recover those messenger RNAs, the tissuemust first be subjected to microdissection. This is very laborious, asonly a small amount of cellular material is recovered and the purity aswell as the quality of the cellular material is compromised.

Post-transcriptional events influence the outcome of protein expressionas significantly as transcriptional events. The regulation oftranscription and post-transcription are generally linked. Altering theexpression of transcriptional activators or repressors has importantconsequences for the development of a cell. Therefore, feedback loopsfollowing translational activation of specific mRNAs may change theprogram of transcription in response to growth or differentiationsignals. DNA arrays are well-suited for profiling the steady-statelevels of mRNA globally (i.e., total mRNA or the “transcriptome”).However, because of post-transcriptional events affecting mRNA stabilityand translation, the expression levels of many cellular proteins do notdirectly correlate with steady-state levels of mRNAs (Gygi et al. (1999)Mol. Cell Biol. 19, 1720-1730; Futcher et al. (1999) Mol. Cell Biol. 19,7357-7368).

Many mRNAs contain sequences that regulate their post-transcriptionalexpression and localization (Richter (1996) in Translational Control,eds. J. W. B Hershey, et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, pp. 481-504). These regulatory elements reside in bothintrons and exons of pre-mRNAs, as well as in both coding and noncodingregions of mature transcripts (Jacobson and Peltz (1996) Annu. Rev.Biochem. 65, 693-739; Wickens et al. (1997) Curr. Opin. Genet. Dev. 7,220-232). One example of a sequence-specific regulatory motif is theAU-rich instability element (ARE) present in the 3′-untranslated regions(UTRs) of early-response gene (ERG) mRNAs, many of which encode proteinsessential for growth and differentiation (Caput et al. (1986) Proc.Natl. Acad. Sci. USA 83, 1670-1674; Shaw and Kamen (1986) Cell 46,659-667; Schiavi et al. (1992) Biochim. Biophys. Acta 1114, 95-106; Chenand Shyu (1995) Trends Biochem. Sci. 20, 465-470). Regulation via theARE is poorly understood, but the mammalian ELAV/Hu proteins have beenshown to bind to ARE sequence elements in vitro and to affectpost-transcriptional mRNA stability and translation in vivo (Jain et al.(1997) Mol. Cell Biol. 17, 954-962; Levy et al. (1998) J. Biol. Chem.273, 6417-6423; Fan and Steitz (1998) EMBO J. 17, 3448-3460; Peng et al.(1998) EMBO J. 17, 3461-3470; Keene (1999) Proc. Natl. Acad. Sci. USA96, 5-7).

In vitro RNA selection methods based upon cellular sequences arereported in Gao et al., Proc. Natl. Acad. Sci USA 90, 11207-11211 (1994)and U.S. Pat. Nos. 5,773,246, 5,525,495 and 5,444,149, all to Keene etal., the disclosures of which are incorporated herein in their entirety.Generally, these methods were intended to identify large numbers ofmRNAs present in messenger RNP (mRNP) complexes, and utilized in vitrobinding and amplification of mRNA sequences from large pools ofnaturally-occurring mRNAs. These studies used proteins (referred to asELAV or Hu proteins) known to bind to AU-rich sequence elements presentin the untranslated regions of cellular mRNAs. These experiments led tothe discovery that mRNAs which are structurally or functionally relatedmay be revealed using multi-targeted RNA binding proteins (i.e., RNAbinding proteins that specifically bind more than one target). SeeLevine, et al., (1994) et al., Molecular and Cellular Biology 13,3494-3504; and King, et al., (1993) Journal of Neuroscience 14,1943-1952; reviewed in Antic and Keene (1997) American Journal of HumanGenetics 61, 273-278 and Keene (1999) Proceedings of the NationalAcademy of Sciences (USA) 96, 5-7. However, these reports are limited toin vitro applications, and do not describe in vivo methods forpartitioning RNA into structural or functional subsets using RNA bindingproteins. Although in vitro methods have been used to determineprotein-RNA interactions, their use has certain limitations. Biochemicalmethods are generally reliable when carefully controlled, butRNA-binding can be problematic because many interactions may be of lowaffinity, low specificity or even artifactual. In order to understandRNA-protein interactions and their functional implications on a globalsystems level it is necessary to find reliable methods to monitormessenger RNP complexes in vivo.

The successful immunoprecipitation of epitope-tagged ELAV/Hu proteinwhich has been transfected into pre-neuronal cells has been reported.See Antic et al., Genes and Development 13, 449-461 (1999). Thisimmunoprecipitation was followed by nucleic acid amplification thatallowed for the identification of a messenger RNA encoding neurofilamentM protein (NF-M).

SUMMARY OF THE INVENTION

The present invention relates to a new, in vivo approach for thedetermination of gene expression that utilizes the flow of geneticinformation through messenger RNA clusters or subsets. Recently, thepractice of examining multiple macromolecular events simultaneously andin parallel with the goal of organizing such information computationallyhas taken the designation “-ome.” Thus, the genome identifies all of thegenes of a cell, while the transcriptome is defined as the messenger RNAcomplement of the genome and the proteome is defined as the proteincomplement of the genome (see FIG. 1). The present inventors havedefined several physically organized subsets of the transcriptome anddefined them as dynamic units of the “ribonome”. As described herein,the ribonome consists of a plurality of distinct subsets of messengerRNAs (mRNAs) that are clustered in the cell due to their associationwith RNA-binding proteins (e.g., regulatory RNA-binding proteins). Byidentifying the mRNA components of a cellular ribonome, the cellulartranscriptome can be broken down into a series of subprofiles thattogether can be used to define the gene expression state of a cell ortissue (see FIG. 2). In combination with, for example, high throughputapproaches and by multiplexing RNA processing assays, the presentinventive methods provide the ability to determine the changes thatoccur in multiple gene transcripts simultaneously.

Accordingly, one aspect of the invention is an in vivo method ofpartitioning endogenous, cellular mRNA-binding protein (mRNP) complexes.The method, in one embodiment, comprises contacting a biological samplethat comprises at least one mRNP complex with a ligand that specificallybinds a component of the mRNP complex. The biological sample may be, forexample, a tissue sample, whole tissue, a whole organ, a cell culture,or a cell extract or lysate. The component of the mRNP complex may be aRNA binding protein, a RNA-associated protein, a nucleic acid associatedwith the mRNP complex including the mRNA itself, or another molecule orcompound (e.g., carbohydrate, lipid, vitamin, etc.) that associates withthe mRNP complex. The ligand may be, for example, an antibody thatspecifically binds the component, a nucleic acid that binds thecomponent (e.g., an antisense molecule, a RNA molecule that binds thecomponent), or any other compound or molecule that binds the componentof the complex. The mRNP complex is then separated by binding the ligand(now bound to the mRNP complex) to a binding molecule that binds theligand. The binding molecule may bind the ligand directly (i.e., may bean antibody specific for the ligand), or may bind the ligand indirectly(i.e., may be an antibody or binding partner for a tag on the ligand).The binding molecule will be attached to a solid support, such as a beador plate or column, as known in the art. Accordingly, the mRNP complexwill be attached to the solid support via the ligand and bindingmolecule. The mRNP complex is then collected by removing it from thesolid support (i.e., the complex is washed off the solid support usingsuitable conditions and solvents).

The identity of the mRNA bound within the mRNP complex may then bedetermined, for example, by separating the mRNA from the complex,reverse transcribing the mRNA into cDNA, and sequencing the cDNA.

In embodiments of the invention, therefore, the mRNP complex may beisolated by direct immunoprecipitation of the mRNP complexes, eitherwith or without epitope tags, or by other biochemical partitioningmethods. For example, other proteins bound to or associated with themRNP complex may be immunoprecipitated in order to recover the mRNPcomplex and subsequently the mRNAs bound within the complex. The skilledartisan will appreciate that embodiments of the inventive method allowfor the identification of a plurality of mRNA complexes simultaneously(i.e., concurrently), sequentially, or in batch-wise fashion.Alternatively, the method may be carried out on one biological sample(or portion thereof) numerous times, the steps of the method beingperformed in a sequential fashion, with each iteration of the methodutilizing a different ligand. In any of the described embodiments, cDNAor genomic microarray grids, for example, may be used to identify mRNAsisolated by the inventive method en masse.

A “subset” of mRNA is defined as a plurality of mRNA transcripts ormessages that specifically bind or associate with a mRNP complex. Inother words, subsets are defined by their ability to bind within or to aparticular mRNP complex. The subset will preferably be a quantitative orqualitative fraction of the total mRNA population of the cell.Furthermore, subsets within subsets of mRNAs may be identified using theinvention. The collection of mRNA subsets for any particular cell ortissue sample is an expression profile, also referred to herein as a“ribonomic profile,” for that cell or tissue. It will be appreciatedthat expression profiles will differ from cell sample to cell sample,depending on the type of cell in the sample (e.g., what species ortissue type the cell is), the differentiation status of the cell, thepathogenicity of the cell (i.e., if the cell is infected or if it isexpressing a deleterious gene, such as an oncogene, or if the cell islacking a particular gene), the specific ligand used to isolate the mRNPcomplex, etc. Thus, the expression profile of a cell may be used as anidentifier for the cell, enabling the artisan to compare and distinguishprofiles of different cells.

Stated otherwise, the ribonomic profile provides a pattern recognitionsubset of the global mRNA profile of the cell. When the growth state ofthe cells changes (i.e., tumorigenesis) or the cell is perturbed by apathogen (i.e., a viral infection), the profile will change, and aperturbation of the ribonome can be detected. If cells are treated withcompounds (i.e., drugs) the ribonomic patterns will show desirable orundesirable alteration. Accordingly, the new method provides methods forevaluating the effect of numerous factors on a cell, including toxicity,aging, apoptosis, pathogenesis and cell differentiation.

The new invention has several advantages over previous methods ofpartitioning RNA. First, partitioning of mRNP complexes may be carriedout in vivo, while previous methods were limited to in vitroapplications. The new method is robust enough such that amplification(e.g., by PCR, or alternatively according to the method of Antic et al.(1999) Genes Dev. 13, 449-461) is not necessary to identify cDNAs ofinterest once they are reverse transcribed from the isolated subset ofmRNAs. The present invention does not require the use of iterativeprocesses, such as those set forth in Gao et al., supra. Finally,quantitative determinations are possible with the present invention if,for example, hybridization is used to analyze the expression profile ofthe cell (e.g., in microarray assays or RNAse protection assays (RPA)).

In certain embodiments, therefore, the present invention advantageouslyallows the artisan to identify, monitor, and quantitate mature genetranscripts en masse in order to determine their localization, activity,stability, and translation into protein components of living cells. Themethods described herein advantageously provide a novel approach tofunctional genomics by providing methods of isolating endogenousmessenger-RNA binding proteins, and methods of identifying the subset ofcellular mRNAs contained in mRNP-complexes, using microarrays or otherknown procedures. In preferred embodiments, the inventive methodprovides a basis for investigating and determining functional mRNAnetworks during growth and differentiation cycles by using mRNA-bindingproteins and other mRNP-associated factors to define mRNA subsets.

It will be appreciated that patterns of mRNA subsets (i.e., expressionprofiles) may be altered in the presence of certain compounds (i.e.,drugs) or under various disease conditions. Accordingly, in certainembodiments the inventive methods are useful for screening compoundsthat may be of therapeutic use, and for finding appropriate gene targetsfor the compounds. In other embodiments, the inventive method is usefulfor determining the disease state of a cell, thus providing means forclassifying or diagnosing the presence or predisposition for disease(e.g., cancer).

Gene expression profiles will also vary between differing cell typespresent in a complex tissue, such as a tumor. Some mRNA binding proteinsare present only in certain tumor cells, and a tumor may comprise morethan one cell type. Gene expression profiling for each cell type withina tumor or tissue may be carried out by making an extract of the tissueand immunoprecipitating cell-type specific components of mRNP complexes(e.g., RNA-binding proteins that are attached to mRNA) directly from theextract (i.e., in vivo). The immunoprecipitated pellets will containmRNAs that are only present in the same cells that contain the attachedor associated component. Thus, in certain embodiments, the inventivemethods may be used to characterize and distinguish the gene expressionprofiles of a plurality of cell types, which cell types may co-exist inthe same complex tissue. This can allow the tumor cells to be profiledin whole tumor extracts without having to analyze mRNA in, for example,the non-tumor stromal cells and blood cells that surround tumor cells.The results of such characterization may be useful in determining, forexample, the proper course of treatment for a patient suffering with atumor, when the choice of treatment depends of the kind of tissue (e.g.,endothelial vascular tissue) present in a tumor.

In another embodiment, the present invention provides methods forisolating and optionally identifying proteins that bind or associatewith a mRNP complex.

Alternatively, and in another embodiment, the inventive method may beused to screen test compounds for their ability to modulate geneexpression in a cell. Such methods are useful for screening putativedrugs that may be used in the treatment and/or prevention of disordersassociated with irregularities in gene expression, including but notlimited to cancer.

The foregoing and other aspects of the present invention are explainedin detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating the flow of genetic information fromthe genome to the proteome, and the intermediate levels represented bythe ribonome and the transcriptome. The transcriptome represents thetotal mRNA complement of the genome, but does not necessarily correlatedirectly with protein production. The processing, transport andtranslation of mRNA occurs in the ribonome, where dynamic regulatorysteps determine the proteomic outcome.

FIG. 2 graphically illustrates a comparison of the total cell mRNA (thetranscriptome) with mRNA that has bound within mRNP complexes to form apart of the ribonome. The microarrays representing mRNP complexescontain discrete and more limited subsets of mRNAs, when compared to thetranscriptome.

FIGS. 3A and 3B illustrate multi-probe RNase protection analysis ofmRNAs associated with mRNP complexes. Messenger RNP complexes from celllysates were immunoprecipitated, and the pelleted RNA was extracted andquantitated by RNase protection. FIGS. 3A and 3B show examples of mMycand mCyc-1 multi-probe template sets, respectively. Lanes: (1)undigested riboprobe (slightly larger than RNase-digested product due toriboprobe plasmid template); (2) total cellular RNA; (3) rabbitpre-bleed serum control; (4) mRNAs extracted from HuB mRNPs; (5) mRNAsextracted from PABP mRNPs. An asterisk (*) denotes mRNA species notdetected in total RNA.

FIG. 4 illustrates ribonome profiling of RNA subsets using DNA arrays.The RNA-protein complexes can be derived from cells of two individuals,species, cell types, treatments, developmental stages, etc. mRNA-proteincomplexes are separated immunoprecipitations of complexes are conducted,probes are reverse-transcribed from the RNA template, and a DNA array ofgenes is probed with each pool of RNP-derived probes to generatesubprofiles of gene expression (10). Subprofiles are then compared bysubtraction or addition to generate an overall picture of geneexpression (20). This Figure depicts the ribonomic concept, in whichdifferent mRNPs are isolated and their associated mRNAs identified usingmicroarrays. The subprofiles (mRNP1, mRNP2, . . . mRNPn) of the totalcell profile are shown as additive. Stacked mRNP subprofiles can eachrepresent individual mRNPs within a single cell type, or can representeach individual cell's transcriptome within a complex tissue or tumor.

FIG. 5 sets forth the results of illustrative Example 4, below, andshows mRNAs associated with mRNP complexes using cDNA arrays. Panels:(A) pre-bleed; (B) HuB mRNP complexes; (C) e1F-4E mRNP complexes; (D)PABP mRNP complexes; (E) total cellular RNA. An example of thespecificity of the procedure is indicated by the differential abundanceof the mRNAs encoding β-actin and ribosomal protein S29 among the mRNPprofiles (arrows a and b, respectively). Other examples of suchspecificity are readily observable with other mRNAs (data not shown).

FIG. 6 sets forth the results of illustrative Example 5, below, andshows a comparison of the mRNA profiles from HuB mRNPs before and aftertreatment with retinoic acid (RA). Panels: (A) mRNAs extracted from HuBmRNPs immunoprecipitated from untreated cells; (B) mRNAs extracted fromHuB mRNPs immunoprecipitated from RA-treated cells; (C) acomputer-generated comparison of panels A and B; (D) mRNAs extractedfrom HuA (HuR) mRNPs immunoprecipitated from untreated cells; (E) mRNAsextracted from HuA mRNPs immunoprecipitated from RA-treated cells; (F) acomputer-generated comparison of panels D and E; (G) total complement ofmRNAs extracted from untreated cellular lysate; (H) total complement ofmRNAs extracted from RA-treated cellular lysate; and (I) acomputer-generated comparison of panels G and H. For panels C, F, and I:green bars indicate mRNAs of approximately equal abundance; red barsrepresent mRNAs from HuB mRNPs that were detectable at four-fold orgreater following RA treatment; blue bars represent mRNAs from HuB mRNPsthat were detectable four-fold or greater in cells before RA treatment.

FIG. 7 is a schematic of ribonomic profiling.

FIG. 8 is a schematic outlining a strategy for the identification of newRNA-binding proteins.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be described more fully with reference tothe accompanying drawings, in which preferred embodiments of theinvention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only, and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety.

Except as otherwise indicated, standard methods may be used for theproduction of cloned genes, expression cassettes, vectors, andtransformed cells and plants according to the present invention. Suchmethods are known to those skilled in the art. See e.g., J. Sambrook etal., Molecular Cloning: A Laboratory Manual Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989); F. M. Ausubel etal., Current Protocols In Molecular Biology (Green PublishingAssociates, Inc. and Wiley-Interscience, New York, 1991).

Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three letter code, in accordance with 37 C.F.R. §1.822 and established usage. See, e.g., Patent1n User Manual, 99-102(November 1990) (U.S. Patent and Trademark Office).

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right. “Nucleic acid sequence” as usedherein refers to an oligonucleotide, nucleotide, or polynucleotide, andfragments thereof, and to DNA or RNA of genomic or synthetic originwhich may be single- or double-stranded and represent the sense orantisense strand. The term “nucleic acid” refers to deoxyribonucleotidesor ribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences, and as wellas the sequence explicitly indicated. Two nucleic acids are “recombined”when sequences from each of the two or more nucleic acids are combinedin a progeny nucleic acid.

The terms “nucleic acid” or “nucleic acid sequence” may also be used inreference to genes, cDNA, and mRNA encoded by a gene. The term “gene” isused broadly to refer to any segment of DNA associated with a biologicalfunction. Thus, genes include coding sequences and/or the regulatorysequences required for their expression. Genes also includenon-expressed DNA segments that, for example, form recognition sequencesfor other proteins. Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information.

As used herein, a nucleic acid molecule may be RNA (the term “RNA”encompassing all ribonucleic acids, including but not limited topre-mRNA, mRNA, rRNA, hnRNA, snRNA and tRNA); DNA; peptide nucleic acid(PNA, as described in, e.g., U.S. Pat. No. 5,539,082 to Nielsen et al.,and U.S. Pat. No. 5,821,060 to Arlinghaus et al.); and the analogs andmodified forms thereof. Preferably, the nucleic acid is RNA, and morepreferably the nucleic acid molecule is messenger RNA (mRNA). Nucleicacid molecules of the present invention may be linear or circular, anentire gene or a fragment thereof, full-length or fragmented/digested,“chimeric” in the sense of comprising more than one kind of nucleicacid, and may be single-stranded or double-stranded. Nucleic acid fromany source may be used in the present invention; that is, nucleic acidsof the present invention include but are not limited to genomic nucleicacid, synthetic nucleic acid, nucleic acid obtained from a plasmid,cDNA, recombinant nucleic acid, and nucleic acid that has been modifiedby known chemical methods, as further described herein. Nucleic acidsmay also be products of in vitro selection experiments (also calledaptamers) and other nucleic acid molecules useful for their ability tobind or be bound by other ligands. See D. Kenan, TIBS 19, 57-64 (1994);L. Gold, et al., Annu. Rev. Biochem. 64, 763-798 (1995); S. E. Osborneand A. D. Ellington, Chem. Rev. 97, 349-370 (1997).

Nucleic acids of the present invention may be obtained from anyorganism, including but not limited to bacteria, viruses, fungi, plantsand animals, with animal nucleic acid being preferred, mammalian nucleicacid being more preferred, and human nucleic acid being most preferred.If desired, the nucleic acid may be amplified according to any of theknown nucleic acid amplification methods that are well-known in the art(e.g., PCR, RT—PCR, QC—PCR, SDA, and the like). Nucleic acids of thepresent invention may be, and preferably are, purified according tomethods known in the art.

As summarized above, the present invention relates to in vivo methodsfor partitioning mRNP complexes from a cell. mRNP complexes of thepresent invention is preferably from a biological sample, such as atissue sample, whole tissue, a whole organ (e.g., an entire brain,liver, kidney, etc.), bodily fluid sample, cell culture, cell lysate,cell extract or the like. In a preferred embodiment, the biologicalsample comprises or is obtained from a population of cells. By a“population of cells” herein is meant at least two cells, with at leastabout 10⁻³ being preferred, at least about 10⁻⁶ being particularlypreferred, and at least about 10⁻⁸ to 10⁻⁹ being especially preferred.The population or sample can contain a mixture of different cell typesfrom either primary or secondary cultures, or from a complex tissue suchas a tumor, or may alternatively contain only a single cell type. In apreferred embodiment, cells that are proliferating are used.Alternatively, non-proliferating cells may be used.

Preferred cell types for use in the invention include, but are notlimited to, mammalian cells, including animal (rodents, equines,bovines, canines felines and primates), and human cells, with humancells being preferred. Cells from non-mammalian animals (e.g., avians,fish, reptiles) and from plants may also be used in the practice of thepresent invention. Cells may be tumor cells from tumors of any type,including breast, skin, lung, cervix, colorectal, and brain/CNS tumors,etc. Additionally, non-cancerous cells from any organ may be used,including liver cells, neurons, muscle cells, and the like.

mRNA is referred to herein interchangeably as a “message” or a“transcript”. A “subset” of mRNA is defined as a plurality of mRNA thatspecifically binds within a particular mRNA binding protein complex(mRNP complex). Thus, subsets are defined by their ability to bindwithin or to a particular mRNP complex. The mRNA subset will preferablybe a fraction of the total mRNA population of the cell.

As summarized above, one aspect of the invention is an in vivo method ofpartitioning endogenous cellular mRNA-binding protein (mRNP) complexes.“Endogenous” is used herein to mean that the mRNP complex forms in acell (i.e., in vivo or in situ). The mRNP complex may form in the cellnaturally, i.e., the components of the mRNP complex naturally occur inthe cell and form the mRNP complex. Alternatively, the mRNP complexforms in a cell, even though one or more components of the complex isintroduced into the cell by, e.g., infection or transformation. Forexample, a mRNP complex endogenously forms in a cell when a RNA-bindingprotein that is a component of the mRNP complex is ectopically expressedin the cell by (for example) transforming the cell or infecting the cellwith an expression vector that carries nucleic acid encoding theprotein, and a mRNP complex in which the protein binds is formed.

The method, in one embodiment, comprises contacting a biological samplethat comprises at least one mRNP complex with a ligand that specificallybinds a component of the mRNP complex. The component of the mRNP complexmay be a RNA binding protein, a RNA-associated protein, a nucleic acidassociated with the mRNP complex including the mRNA itself, or anothermolecule or compound (e.g., carbohydrate, lipid, vitamin, etc.) thatassociates with the mRNP complex. A component “associates” with a MRNPcomplex if it binds or otherwise attaches to the mRNP complex with a Kdof about 10⁻⁶ to about 10⁻⁹. In a preferred embodiment, the componentassociates with the complex with a Kd of about 10⁻⁷ to about 10⁻⁹. In amore preferred embodiment, the component associates with the complexwith a Kd of about 10⁻⁸ to about 10⁻⁹.

The ligand may be any molecule that specifically binds the component ofthe mRNP complex. For example, the ligand may an antibody thatspecifically binds the component, a nucleic acid that binds thecomponent (e.g., an antisense molecule, a RNA molecule that binds thecomponent), or any other compound or molecule that specifically bindsthe component of the complex. In certain embodiments, the ligand may beobtained by using the serum of a subject (i.e., a human or animalsubject) that has a disorder known to be associated with the productionof mRNP-complex specific antibodies or proteins. Examples of thesedisorders include autoimmune disorders such as systemic lupuserythematosus (“lupus” or SLE) and a number of cancers. In certainembodiments, the ligand may be “tagged” with another compound ormolecule in order to facilitate the separation, observation or detectionof the ligand. In one embodiment of the invention, the ligand is“epitope tagged,” as described in the art. Suitable tags are known inthe art and include but are not limited to biotin, the MS2 proteinbinding site sequence, the U1snRNA 70k binding site sequence, theU1snRNA A binding site sequence, the g10 binding site sequence(commercially available from Novagen, Inc., Madison, Wis., USA), andFLAG-TAG® (Sigma Chemical, St. Louis, Mo., USA).

The mRNP complex is then separated by binding the ligand (now bound tothe mRNP complex) to a binding molecule that specifically binds theligand. The binding molecule may bind the ligand directly (i.e., may bean antibody or protein specific for the ligand), or may bind the ligandindirectly (i.e., may be an antibody or binding partner for a tag on theligand). Suitable binding molecules include but are not limited toprotein A, protein G, streptavidin. Binding molecules may also beobtained by using the serum of a subject suffering from, for example, anautoimmune disorder or cancer. In certain embodiments, the ligand is anantibody that binds the component of the mRNP complex via the Fab regionof the antibody, and the binding molecule in turn binds the Fc region ofthe antibody. The binding molecule will be attached to a solid support,such as a bead, well, pin, plate or column, as known in the art.Accordingly, the mRNP complex will be attached to the solid support viathe ligand and binding molecule.

The mRNP complex is then collected by removing it from the solid support(i.e., the complex is washed off the solid support under appropriatestringency conditions, using suitable solvents that may be determined byskilled artisans).

In certain embodiments of the invention, the mRNP complex may bestabilized by cross-linking prior to binding the ligand thereto.Cross-linking, as used herein, means covalently binding (e.g.,covalently binding the components of the mRNP complex together).Cross-linking may be contrasted with ligand-target binding, or bindingmolecule-ligand binding, which is generally non-covalent binding.Cross-linking may be carried out by physical means (e.g., by heat orultraviolet radiation), or chemical means (e.g., by contacting thecomplex with formaldehyde, paraformaldehyde, or other knowncross-linking agents), which means are known or determinable by thoseskilled in the art. In other embodiments, the ligand may be cross-linkedto the mRNP complex after binding the mRNP complex. In additionalembodiments, the binding molecule may be cross-linked to the ligandafter binding to the ligand. In yet other embodiments, the bindingmolecule may be cross-linked to the solid support.

The skilled artisan will appreciate the inventive method allows for theidentification of a plurality of mRNP complexes simultaneously (e.g.,“en masse”). For example, a biological sample may be contacted with aplurality of ligands specific for different mRNP complex components. Aplurality of mRNP complexes from the sample will bind the variousligands. The plurality of mRNP complexes can then be separated usingappropriate binding molecules, thus isolating the plurality of mRNPcomplexes. The mRNP complexes and the mRNAs contained within thecomplexes may then be characterized and/or identified by methodsdescribed herein and known in the art. Alternatively, the method may becarried out on one sample numerous times, the inventive steps beingperformed in a sequential fashion, with each iteration of stepsutilizing a different ligand.

As set forth above, a subset of mRNA identifies a pattern-recognitionprofile that is characteristic of the RNA structural or functionalnetworks in that sample. The collection of mRNA subsets for anyparticular cell or tissue sample constitutes a gene expression profile,and more specifically a ribonomic gene expression profile, for that cellor tissue. It will be appreciated that ribonomic expression profiles maydiffer from cell to cell, depending on the type of cell in the sample(e.g., what species or tissue type the cell is), the differentiationstatus of the cell, the viability of the cell (i.e., if the cell isinfected or if it is expressing a deleterious gene, such as an oncogene,or if the cell is lacking a particular gene or not expressing aparticular gene), the specific ligands used to isolate the mRNPcomplexes, etc. Thus, the ribonomic expression profile of a cell may beused as an identifier for the cell, enabling the artisan to compare anddistinguish profiles or subprofiles of different cells. The genesidentified by the RNAs present in each ribonomic pattern form distinctsubsets that may be associated with a particular cell cycle, stage ofdifferentiation, apoptosis or stress induction, viral infection, orcancer.

cDNAs may be used to identify mRNP complexes partitioned with a ligandor ligands specific for a component of the mRNP complex. cDNA microarraygrids, for example, may be used to identify mRNA subsets en masse.Microarrays are precisely aligned grids in which each target nucleicacid (e.g., gene) has a position in a matrix of carefully spotted cDNAs.See Gerhold et al., supra, Duggan et al., supra, and Brown et al.,supra. Alternatively, genomic microarrays (e.g., microarrays wherein thetarget nucleic acids may contain introns and exons) may be used.Therefore, each gene or target nucleic acid being examined on amicroarray has a precise address that can be located, and the bindingcan be quantitated. Microarrays in the form of siliconized chips orthose based upon cDNA blots on nylon or nitrocellulose are commerciallyavailable. Glass slides can also be customized with oligonucleotides orDNAs for detection of complementary RNA sequences. In all of thesecases, the hybridization platforms allow identification of the mRNAs ina sample based upon the stringency of binding and washing. This has beenreferred to as “sequencing by hybridization.” Although microarraytechnology is one method of analysis, it is only one way to identifyand/or sequence the mRNAs in the mRNA subset. Alternative approachesinclude but are not limited to differential display, phagedisplay/analysis, SAGE or simply preparing cDNA libraries from the mRNApreparation and sequencing all members of the library.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

In a preferred embodiment, amplification of the mRNA isolated accordingto the present invention, and/or the cDNA obtained from the mRNA is notcarried out during the identification of the nucleic acid, and is notnecessary or required by the present invention. However, the skilledartisan may choose to amplify the nucleic acid that is the subject ofidentification (e.g., the nucleic acid being identified via microarrayanalysis and/or sequencing) for convenience, as a matter of preference,and/or to comply with the specification/instructions of certaincommercially available microarrays or microarray analysis systems. Thus,if desired, the nucleic acid may be amplified according to any of thenumerous known nucleic acid amplification methods that are well-known inthe art (e.g., PCR, RT—PCR, QC—PCR, SDA, and the like).

Methods of the present invention may be carried out in several ways,according to the needs of the practitioner and the purpose for which theinvention is carried out. For example, in one embodiment, mRNA-bindingprotein complexes that are unique to a cell type of interest areidentified. In an example of such an embodiment, an antibody that isspecific for the mRNP complex can be used to immunoprecipitate thecomplex with its associated mRNAs. The RNAs may then identified to formthe ribonomic expression profile of that cell type, or alternatively maybe isolated for (as an example) drug screening. The mRNA candidates forpost-transcriptional regulation may be analyzed en masse, as a subset,for changes in mRNA stability during the cell cycle or developmentalevents. In certain embodiments, the methods may be carried out byisolating nuclei from cells undergoing developmental or cell cyclechanges, performing nuclear run-off assays according to known techniquesto obtain transcribing mRNAs, and then comparing the transcribing mRNAswith the global mRNA levels in the same cells using CDNA microarrays.These methods thus provide the ability to distinguish transcriptionalfrom post-transcriptional effects on steady state mRNA levels en masse.

In another embodiment, cells in culture are transformed to express aRNA-binding protein (RBP) or RNA-associated protein (RAP) that willassociate with particular mRNAs only in a cell type of interest. DNAencoding the RBP or RAP may be carried by a recombinant vector (e.g., aplasmid, a viral vector) and transformed into the cell by known means,after which the RBP or RAP is expressed in the cell. Any RBP or RAP canbe used, as described further herein. The protein may be in its nativeform, or it may be tagged (e.g., epitope tagged) for easy recovery fromthe cell. Detection of multiple RNA targets in vivo that are bound orassociated with RBPs or RAPs may be carried out by using accessibleepitopes, if necessary, but preferably is carried out without tags. Incases where the epitopes on the RBPs or RAPs are inaccessible orobscured, epitope tags on ectopically expressed recombinant proteins maybe used. The transformed cell may be mixed with other cell types or maybe implanted in an animal or human subject. A ligand (e.g., an antibody)that is specific for the protein can used to immunoprecipitate theprotein with its associated messenger RNAs from an extract of a tissuecontaining the transformed cell. The mRNA complexes and its associatedRNAs may then identified to form the expression profile of that celltype or is otherwise analyzed (e.g., for drug development).

In still another embodiment, a specific cell type in an animal isengineered with one or more cell-type specific gene promoters to expressa RBP or RAP in the cell type of interest. As set forth above, the genepromoter and the RBP or RAP may be carried on one or more vectors andtransformed into the cell, where the RBP or RAP is expressed. In oneembodiment, a ligand (e.g., an antibody) that is specific for thisprotein can used to immunoprecipitate the protein with its attached orassociated messenger RNAs from an extract of a tissue containing thecell type of interest. The RNAs are then identified to form theexpression profile of that cell type or isolated, e.g., for drugdevelopment.

RNA binding proteins (RBPs) and RNA-associated proteins (RAPs) useful inthe practice of the present invention are known in the art, or mayalternatively be identified and discovered by methods described herein.RNA binding proteins are now known to be involved in the control of avariety of cellular regulatory and developmental processes, such as RNAprocessing and compartmentalization, RNA stabilization, mRNA translationand viral gene expression. RNA binding proteins include poly A-bindingprotein (“PABP,” which gives rise to a subset of the total mRNApopulation that is quantitatively different from the total mRNApopulation), and other general RNA binding proteins, as well asRNA-binding proteins that are attached to only one or a few messengerRNAs in a particular cell type. Other useful proteins are autoantibodiesreactive with RNA and RNA-binding proteins.

Examples of useful RNA binding proteins include the four ELAV/Humammalian homologues of the Drosophila ELAV RNA-binding protein (Good(1995) Proc. Natl. Acad. Sci. USA 92, 4557-4561; Antic and Keene, supra.HuA (HuR) is ubiquitously expressed while HuB, HuC and HuD (and theirrespective alternatively-spliced isoforms) are predominantly found inneuronal tissue, but can also be expressed as tumor cell-specificantigens in some small cell carcinomas, neuroblastomas, andmedulloblastomas (reviewed in Keene (1999) Proc. Natl. Acad. Sci. USA96, 5-7). All Hu proteins contain three RNA-recognition motifs (RRMs),which confer their binding specificity for AREs (Antic and Keene, supra;Kenan et al. (1991) Trends Biochem. Sci. 16, 214-220; Burd and Dreyfuss(1994) Science 265, 615-621). The evidence for ARE binding by Huproteins began with the identification of an AU-rich binding consensussequence from a randomized combinatorial RNA library that was screenedwith recombinant HuB (Levine et al. (1993) Mol. Cell Biol. 13,3494-3504; Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91,11207-11211). These and other studies demonstrated that Hu proteins bindin vitro to several ARE-containing ERG mRNAs including c-myc, c-fos,GM-CSF and GAP-43 (Levine et al. (1993) Mol. Cell Biol. 13, 3494-3504;Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11207-11211; King etal. (1994) J. Neurosci. 14, 1943-1952; Liu et a. (1995) Neurology 45,544-550; Ma et al (1996) J. Biol. Chem. 271, 8144-8151; Abe et al.(1996) Nucleic Acids Res. 24, 2011-2016; Chung et al. (1997) J. Biol.Chem. 272, 6593-6598; Fan and Steitz (1998) EMBO J. 17, 3448-3460; Anticet al. (1999) Genes Dev. 13, 449-461).

The binding of Hu proteins to ARE-containing mRNAs can result in thestabilization and increased translatability of the mRNA transcripts(Jain et al. (1997) Mol. Cell Biol. 17, 954-962; Levy et al. (1998) J.Biol. Chem. 273, 6417-6423; Fan and Steitz (1998) EMBO J. 17, 3448-3460;Peng et al. (1998) EMBO J. 17, 3461-3470). The neuron-specific Huproteins are one of the earliest neuronal markers produced interatocarcinoma cells following retinoic acid (RA)-treatment to induceneuronal differentiation (Antic et al., supra; Gao and Keene (1996) J.Cell Sci. 109, 579-589).

In one embodiment, the ligand used to carry out the invention is a RNAbinding protein selected from the RNA Recognition Motif (RRM) family ofcellular proteins involved in pre-messenger RNA processing. One exampleof such a protein is the U1A snRNP protein. More than 200 members of theRRM superfamily have been reported to date, the majority of which areubiquitously expressed and conserved in phylogeny (Query et al, Cell(1989) 57: 89-101; Kenan et al, Trends Biochem. Sci. (1991)16: 214-220).Most are known to have binding specificity for polyadenylate mRNA orsmall nuclear ribonucleic acids (e.g. U1, U2, etc.) transfer RNAs, 5S or7S RNAs. They include but are not limited to hnRNP proteins (A, B, C, D,F, F, G, H, I, K, L), RRM proteins CArG, DT-7, PTB, K1, K2, K3, HuD,HUC, rbp9, eIF4B, sx1, tra-2, AUBF, AUF, 32KD protein, ASF/SF2, U2AF,SC35, and other hnRNP proteins. Tissue-specific members of the RRMfamily are less common, including IMP, Bruno, AZP-RRMI, X16 which isexpressed in pre-B cells, Bj6 which is a puff-specific Drosophilaprotein and ELAV/Hu, which are neuron specific.

RNA-binding and RNA-associated proteins useful in the practice of thepresent invention include those isolated using autoimmune and cancerpatient sera. A non-comprehensive list of RNA-binding and RNA-associatedproteins useful in the practice of the present inventions is set forthbelow in Table 1.

TABLE 1 RNA Binding and RNA Associated Proteins SLBP DAN TTP Hel-N 1Hel-N2 el F-4A eIF-4B eIF-4G eIF-4E eIF-5 eIF-4EBP MNK1 PABP p62 KOC p90La Sm Ro U1-70K AUF-1 RNAse-L GAPDH GRSF Ribosomal Po, P1, P2/L32 PM-SclFMR Stauffen Crab 95 TIA-1 Upf1 RNA BP1 RNA BP2 RNA BP3 CstF-50 NOVA-1NOVA-2 CPEBP GRBP SXL SC35 U2AF I ASF/SF2 ETR-1 IMP-1 IMP-2 IMP-3 ZBPLRBP-1 Barb PTB uPAmRNA BP BARB1 BARB2 GIFASBP CYP mRNA BP IRE-BP p50RHA FN mRNA BP AUF-1 GA mRNA BP Vigillin ERBP CRD-BP HuA HuB HuC HuDhnRNP A hnRNP B hnRNP C hnRNP D hnRNP E hnRNP F hnRNP G hnRNP H hnRNP KhnRNP L U2AF

The identification of new (i.e., novel, previously unknown) RNA-bindingproteins (RBPS) and RNA associated proteins, is another aspect of theinvention. Thus, in one embodiment of the invention, a RNA of interest(depicted in FIG. 8 as “RNA Y”) is used as a “bait” to trap a new RBP orRAP. In a preferred embodiment, RNA Y is first converted to a cDNA usingstandard molecular biology techniques and is subsequently ligated at the3′ or 5′ end to another fragment of DNA (referred to herein as the“tagging DNA”) that encodes a sequence (e.g., a RNA) that will bind aligand of the present invention (the ligand being illustrated as protein“X” in FIG. 8). In other words, the tagging DNA encodes a bindingpartner of the ligand. Useful ligands may, in some embodiments, beobtained from (i.e., by using) the serum of a subject (i.e., a human oranimal subject) that has a disorder known to be associated with theproduction of mRNP-complex specific antibodies or proteins, includingautoimmune disorders and cancer. Useful binding partners includeantibodies to the ligand.

The resulting DNA chimera is fused to a promoter in an expression vector(e.g., a plasmid) and expressed in living cells (e.g., in a cellculture) to produce a RNA fusion molecule. In an alternative embodiment,the expression vector is infected into the cells by a virus, preferablya recombinant virus. A cell-free extract from the culture is preparedand contacted with the ligand (e.g., protein X) which has beenimmobilized on a solid support. After an incubation period, the ligandand the attached/associated RNA fusion molecule and its associated RBPsor RAPs are washed to remove residual cellular material. After the washstep, the RBPs or RAPs are removed from the RNA-protein complex andanalyzed (e.g., sequenced using standard methods of microsequencing).

Once partial protein sequence is obtained, the corresponding gene may beidentified from known databases containing cDNA and genomic sequences.Preferably, the gene is isolated, the protein is expressed, and anantibody is generated against the recombinant protein using knowntechniques. The antibodies are then used to recover and confirm theidentity of the endogenous RBP or RAP. Subsequently, the antibody can beused for ribonomic analysis (see examples below) to determine the subsetof cellular RNAs that cluster with (i.e., associate with) RNA Y.Furthermore, the RBP or RAP may be tested for its ability to regulatethe translation of the protein encoded by RNA Y, and may be tested forvalidation as a drug target. Likewise, proteins encoded by the cellularRNAs that cluster with RNA Y may be tested for validation as drugtargets, as further described herein.

Antibodies that specifically bind mRNP complexes are thus an aspect ofthe invention. Antibodies to mRNP complexes may be generated usingmethods that are well known in the art. Such antibodies may include, butare not limited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments, and fragments produced by a Fab expression library.Antibodies and fragments thereof may also be generated using antibodyphage expression display techniques, which are known in the art.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith the mRNP complex or any fragment or component thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase an immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (BacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

Monoclonal antibodies to the components of the mRNP complex may beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al.(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030;Cole, S. P. et al. (1984) Mol. Cell Biol. 62.109-120). Briefly, theprocedure is as follows: an animal is immunized with the mRNP complex orimmunogenic fragments or conjugates thereof. Lymphoid cells (e.g.splenic lymphocytes) are then obtained from the immunized animal andfused with immortalizing cells (e.g. myeloma or heteromyeloma) toproduce hybrid cells. The hybrid cells are screened to identify thosewhich produce the desired antibody.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al., Proc. Natl. Acad. Sci. 86, 3833-3837 (1989));Winter, G. et al., (1991) Nature 349, 293-299 (1991)).

Antibody fragments that contain specific binding sites for mRNPcomplexes may also be generated. For example, such fragments include,but are not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity for the mRNP. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays will typically involve the measurement ofcomplex formation between the component of the mRNP complex and itsspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes ispreferred, but a competitive binding assay may also be employed.

Kits or devices (e.g., fluidic devices) containing columns in whichantibodies to various mRNP complexes or components thereof (e.g.,antibodies to RNA-binding proteins) are immobilized are another aspectof the invention. The antibodies may be conjugated to a solid supportsuitable for a diagnostic assay (e.g., beads, plates, slides or wellsformed from materials such as latex or polystyrene) in accordance withknown techniques, such as precipitation. Antibodies may likewise beconjugated to detectable groups such as radiolabels (e.g. ³⁵S, ¹²⁵I,¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein) in accordancewith known techniques. Devices of the present invention will preferablyinclude at least one reagent specific for detecting the binding betweenan antibody to a mRNA-binding protein and the protein itself. Thereagents may also include ancillary agents such as buffering agents andprotein stabilizing agents, e.g., polysaccharides and the like. Thedevice may further include, where necessary, agents for reducingbackground interference in a test, control reagents, apparatus forconducting a test, and the like. The device may be packaged in anysuitable manner, typically with all elements in a single container alongwith a sheet of printed instructions for carrying out the test.

Certain embodiments of the invention relate to methods of screening testcompounds for therapeutic, diagnostic or pharmaceutical use, based uponeach compound's effect on the ribonomic profile of a cell or tissuesample. In an example of such an embodiment, cells are grown underconditions where the cells have no contact with the test compound (i.e.,the cells are not treated). A ribonomic profile of the cell type is thenproduced, and the mRNA subsets identified. The ribonomic profile of thenon-treated cells is then compared to a ribonomic profile of the samecell type that has been treated with the test compound. Any differencebetween the two profiles is an indication that the test compound has aneffect (directly or indirectly) on the expression of certain genes ofthe cell, and may be an indication that the test compound is a candidatefor therapeutic or diagnostic use. Alternatively, the ability of acompound to effect gene expression may identify the gene as a target forfurther testing. A “difference” in the profiles refers to any modulationor change in expression between the two profiles. “Modulation” can referto an increase in expression, a decrease in expression, a change in thetype or kind of expression present, a complete cessation of expression(i.e., an absence of expression), or the instigation of expression.Suitable compounds that may be used include but are not limited toproteins, nucleic acids, small molecules, hormones, antibodies,peptides, antigens, cytokines, growth factors, pharmacological agentsincluding chemotherapeutics, carcinogenics, or other cells (i.e.cell-cell contacts). Cells may also be screened for the effects ofenvironmental or physiological factors such as radiation, actionpotentials, etc. on normal gene expression.

In another embodiment of the invention, an mRNP component itself, itscatalytic or immunogenic fragments or oligopeptides thereof, can be usedfor screening libraries of compounds in any of a variety of drugscreening techniques. The fragment employed in such screening may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The binding between the mRNP complex and thecompound being tested may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In one embodiment, as applied to the mRNP complex, aplurality of different test compounds are synthesized on or affixed to asolid substrate, such as plastic pins or some other surface. The testcompounds are reacted with the mRNP complex, or fragments and/orcomponents thereof, and washed. The bound mRNP complex or componentthereof is then detected by methods well known in the art.

In summary, the present invention provides powerful in vivo methods fordetermining the ribonomic profile of a cell and detecting changes in thesame. The invention has numerous uses, including but not limited to themonitoring of tumor development, state of growth or state ofdevelopment, perturbations of a biological system such as disease, drugor toxin treatment, and the state of cell aging or death. The inventionalso finds use in distinguishing ribonomic profiles amongst organismssuch as plant, fungal, bacterial, viral, protozoan, or animal species.

The present invention can be used to discriminate betweentranscriptional and post-transcriptional contributions to geneexpression and to track the movement of RNAs through RNP complexes,including the interactions of combinations of proteins with RNAs in RNPcomplexes. Accordingly, the present invention can be used to study theregulation of stability of RNAs. The present invention can be used toinvestigate the activation of translation of mRNAs as single or multiplespecies by tracking the recruitment of mRNAs to active polysomes,measuring the sequential, ordered expression of mRNAs, and measuring thesimultaneous, coordinate expression of multiple mRNAs. The presentinvention can also be used to determine the trans-acting functions ofRNAs themselves upon contacting other cellular components. These andnumerous other uses will be made apparent to the skilled artisan uponstudy of the present specification and claims,

The following Examples are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 RNase Protection in a Multiprobe System: Materials and Methods

It has previously been reported that HuB (Hel-N1) immunoprecipitation,using a g10 epitope tag, resulted in the co-immunoprecipitation of amRNA, which once amplified by RT-PCR and sequenced, was found to encodeNF-M protein (Antic, 1999, supra). In this example, the same approach isexpanded to using a multiprobe RNase protection assay to rapidlyoptimize the immunoprecipitation of several endogenous mRNA-protein(mRNP) complexes containing different mRNA-binding proteins. In themulti-probe system, many mRNAs, from mRNP pellets, can be assayed in asingle lane of polyacrylamide gel.

Cell Culture and Transformation. Murine P19 embryonal carcinoma cellswere obtained from the ATCC and maintained in monolayer culture using.alpha.-MEM without phenol red (Gibco-BRL 41061-029) supplemented with7.5% Bovine Calf Serum, 2.5% Fetal Bovine Serum (Hyclone) and 100 UPenicillin/Streptomycin. Cells were grown in tissue culture flasks orplates that had been pre-coated with 0.1% gelatin (Sigma Chemicals) thatwas removed prior to use. Monolayer cell cultures were maintained in 5%CO₂ at 37° C.

P19 cells were stably transfected with a SV40 promoter-drivenpAlpha2-gene 10-HuB plasmid that ectopically expressed a gene 10-taggedneuron-specific HuB protein termed He1-N2 (Gao et al. (1994) Proc. Natl.Acad. Sci. USA 91, 11207-11211). The transfected plasmid was maintainedby supplementing the medium with 0.2 mg/ml G418 (Sigma Chemicals).Although it lacks thirteen amino acids from the hinge region connectingRNA-recognition motifs (RRMs) 2 and 3 of He1-N1, the RRMs are identicaland in vitro binding experiments have indicated no differences in theAU-rich RNA binding properties of He1-N1 and He1-N2 (Gao et al. (1994)Proc. Natl. Acad. Sci. USA 91, 11207-11211; Abe et al. (1996) NucleicAcids Res. 24, 2011-2016; unpublished results).

Antibodies. Monoclonal anti-gene 10 (g10) antibodies were produced aspreviously described (Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91,11207-11211; Antic et al. (1999) Genes Dev. 13, 449-461). Polyclonalsera reactive with HuA were produced as previously described (Levine etal. (1993) Mol. Cell. Biol. 13, 3494-3504; Atasoy et al. (1998) J. CellSci. 111, 3145-3156). Antibodies reactive with Poly A binding protein(PABP) were kindly provided by Dr. N. Sonenberg of McGill University(Canada).

Preparation of Cell-Free Extracts. Cells were removed from tissueculture plates with a rubber scraper and washed with cold PBS. The cellswere resuspended in approximately two pellet volumes of polysome lysisbuffer (PLB) containing 100 mM KCl, 5 mM MgCL₂, 10 mM HEPES pH 7.0, and0.5% NP-40 with 1 mM DTT, 100 U/mL RNase OUT (GIBCO-BRL), 0.2% vanadylribonucleoside complex (VRC)(GIBCO-BRL), 0.2 mM PMSF, 1 mg/mL pepstatinA, 5 mg/mL bestatin, and 20 mg/mL leupeptin added fresh at the time ofuse. The lysed cells were then frozen and stored at −100° C. At the timeof use, the cell lysate was thawed and centrifuged at 12,000 rpm in atabletop microfuge for 10 minutes at 4° C. The supernatant was removedand centrifuged a second time at 16,000 rpm in a tabletop microfuge for5 minutes at 4° C. before being stored on ice or refrozen at −100° C.The mRNP cell lysate contained approximately 30-50 mg/mL total protein.

Immunoprecipitations. For immunoprecipitation, Protein A sepharose beads(Sigma Biochemicals) were swollen 1:5 v/v in NT2 buffer (50 mM Tris pH7.4, 150 mM NaCl, 1 mM MgCl₂, and 0.05% NP-40) supplemented with 5%bovine serum albumin. A 300 μL aliquot of 1:5 v/v pre-swollen Protein Abead slurry was used per immunoprecipitation reaction and incubatedovernight at 4° C. with excess immunoprecipitating antibody (typically5-20 L, depending on the reagent). The antibody-coated Protein A beadswere washed 5 times with ice-cold NT2 buffer and resuspended in 900 μLof NT2 buffer supplemented with 100 U/mL RNase OUT, 0.2% VRC, 1 mM DTT,and 20 mM EDTA. The beads were briefly vortexed and 100 μL of the mRNPcell lysate was added. The beads were immediately centrifuged and 100 μLof the supernatant was removed to represent total cell mRNP lysate(essentially one-tenth the quantity of lysate used in the mRNPimmunoprecipitations). The immunoprecipitation reaction and an aliquotremoved to represent total cell mRNP lysate were tumbled at roomtemperature for a time period of from zero time to up to two hours.Following incubation, the Protein A beads were washed four times withice-cold NT2 buffer followed by two washed with NT2 buffer supplementedwith 1 M urea. Washed beads were resuspended in 100 μL NT2 buffersupplemented with 0.1% SDS and 30 μg proteinase K and incubated for 30minutes in a 55° C. water bath. Following proteinase K digestion,immunoprecipitated RNA was isolated with two phenol/chloroform/isoamylalcohol extractions and ethanol precipitated.

RNase Protection Assays. After mRNP complexes were immunoprecipitatedfrom cell lysates and the bound RNA extracted, it was assayed by RNaseprotection using the PharMingen Riboquant assay (San Diego, Calif.)according to the manufacturer's instructions (45014K). Briefly,extracted RNA was hybridized with excess ³²P-labeled riboprobesgenerated from templates specific for mRNAs encoding L32, GAPDH, severalmurine Myc-related proteins (template set 45356P) and cyclins (templateset 45620P). Non-duplexed RNA was digested by treatment with RNase A+T1.The resulting fragments were resolved by denaturing polyacrylamide/ureagel electrophoresis. Because the length of the riboprobe for each mRNAspecies was a unique size, all detectable mRNA species in a sample couldbe resolved in a single gel lane. Protected riboprobe fragments werevisualized on a phosphoimaging screen (Molecular Dynamics) after 24hours of exposure. Phosphoimages were scanned using the MolecularDynamics STORM 860 System at 100 micron resolution and analyzed usingMolecular Dynamics ImageQuant Software (V 1.1).

EXAMPLE 2 RNase Protection in a Multiprobe System: Experimental Results

FIG. 3 shows an immunoprecipitation of HuB and Poly-A binding protein(PABP)-mRNP complexes from extracts of murine P19 cells stablytransfected with g10-HuB cDNA. No mRNAs were detected in pelletsimmunoprecipitated with polyclonal pre-bleed rabbit sera (FIGS. 3A and3B, lane 3), or with many other rabbit, mouse, and normal human seratested with this assay (data not shown). The profiles of mRNAsassociated with HuB mRNP complexes included n-myc, l-myc, b-myc, max andcylins A2, B1, C, D1, and D2, but not sin3, cyclin D3, cyclin B2, L32 orGAPDH mRNAs (FIGS. 3A and 3B, lane 4). In contrast, the profiles ofmRNAs extracted from PABP-mRNP complexes resembled the profiles of totalRNA, but showed enriched levels of L32 and GAPDH and decreased levels ofsin3 mRNA (FIGS. 3A and 3B, lane 5). It was concluded that antibodiesreactive with these cellular RNA-binding proteins could be used toimmunoprecipitate mRNP complexes and to recover mRNAs with which theyare specifically associated. These results are consistent with thepostulate role of Hu proteins in regulating post-transcriptional geneexpression during cell growth and differentiation. (Jain et al. (1997)Mol. Cell. Biol. 17, 954-962; Levy et al. (1998) J. Biol. Chem. 273,6417-6423; Fan and Steitz (1998) EMBO J. 17, 3448-3460; Peng et al.(1998) EMBO J. 17, 3461-3470; Antic and Keene (1997) Am. J. Hum. Genet.61, 273-278; Levine et al. (1993) Mol. Cell Biol. 13, 3494-3504; Gao etal. (1994) Proc. Natl. Acad. Sci. USA/91, 11207-11211; King et al.(1994) J. Neurosci. 14, 1943-1952; Liu et al. (1995) Neurology 45,544-550; Ma et al. (1996) J. Biol. Chem. 271, 8144-8151; Abe et al.(1996) Nucl. Acids Res. 24, 2011-2016; Antic et al. (1999) Genes Dev.13, 449-461; Chung et al. (1997) J. Biol. Chem. 272, 6593-6598; Akamatsuet al. (1999) Proc. Natl. Acad. Sci. USA 96, 9884-9890; Sachs et al.(1997) Cell 89, 831-838; Aranda-Abreu et al. (1999) J. Neurosci. 19,6907-6917).

EXAMPLE 3 Identification of mRNA Subsets Associated with RNA BindingProteins En Masse Using cDNA Arrays: Materials and Methods

To further expand the ability to identify the mRNAs associated inendogenous mRNP complexes, this example describes the use of a cDNAarray filter as a highly specific and sensitive method to detect a mRNAsubset without amplification or iterative selection (FIG. 4).

Antibodies. Monoclonal anti-gene 10 (g10) antibodies were produced aspreviously described (see D. Tsai et al., Proc. Natl. Acad. Sci. USA,89, 8864-8868 (1992); Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91,11207-11211; Antic et al. (1999) Genes Dev. 13, 449-461). Polyclonalsera reactive with Hu proteins were produced as previously described(Levine et al. (1993) Mol. Cell. Biol. 13, 3494-3504; Atasoy et al.(1998) J. Cell Sci. 111, 3145-3156). Antibody against 5′ cap bindingprotein (e1F-4E) was obtained from Transduction Laboratories (San Diego,Calif.). Antibodies reactive with Poly A binding protein (PABP) werekindly provided by Dr. N. Sonenberg of McGill University (Canada).

Cell Culture and Differentiation. Preparation of transgenic cells was asdescribed in Example 1. Chemical treatment with retinoic acid (RA) wasused to induce neuronal differentiation by treating 5×10⁵ P19 cells,placed a 60 mm petri dish (Fisher Scientific, Pittsburgh, Pa., USA,Number 8-757-13A), with 0.5 μM RA (Sigma Chemicals, St. Louis, Mo., USA,Number R2625), as described in Gao and Keene (1996)). After two days,25% of the cells that had formed into clumps were removed, placed in newpetri dishes, and supplemented with fresh medium and RA. Following anadditional two days, cell aggregates were washed once withphosphate-buffered saline (PBS) and trypsinized. The cells were thenplated into two 100 mm gelatin-coated tissue culture plates. Cells wereharvested after an additional four days. The RA treated HuB (He1-N2)stably transfected P19 cells grew neurites and displayed characteristicsneuronal markers and morphology, but did not terminally differentiateand remained susceptible to killing with mitotic inhibitors. Cell-freeextracts and immunoprecipitations were as described in Example 1.

cDNA Array Analysis. cDNA array analysis was performed using Atlas™Mouse Arrays (Clontech, Inc., Palo Alto, Calif.) that contain a total of597 cDNA segments spotted in duplicate, side-by-side on a nylonmembrane. Probing of cDNA arrays was performed as described in theClontech Atlas™ cDNA Expression Arrays User Manual (PT3140-1). Briefly,RNA was extracted from HuB stably transfected P19 embryonal carcinomacells and used to produce reverse transcribed probes. A pooled set ofprimers, complementary to the genes represented on the array, was usedfor the reverse transcription probe synthesis, which was radiolabeledwith ³²p α-dATP. The radiolabeled probe was purified by passage overCHROMA SPIN™-200 columns (Clontech, Inc., Palo Alto, Calif.) andincubated overnight with an array membrane using ExpressHyb™hybridization solution (Clontech, Inc., Palo Alto, Calif.). Followinghybridization, the array membrane was washed and visualized on aphosphorimaging screen (Molecular Dynamics, Sunnyvale, Calif., USA).

Phosphorimages were scanned using the Molecular Dynamics STORM 860System at 100 micron resolution and stored as files. Images wereanalyzed using AtlasImage™. 1.0 and 1.01 software (Clontech, Inc., PaloAlto, Calif.). The signal for any given gene was calculated as theaverage of the signals from the two duplicate cDNA spots. As describedin the AtlasImage™. 1.0 software manual (Clontech, Inc., Palo Alto,Calif.), a default external background setting was used in conjunctionwith a background-based signal threshold to determine gene signalsignificance. The signal for a gene was considered significantly abovebackground if its adjusted intensity (total signal minus background) wasmore then two-fold the background signal. Comparisons of multiple cDNAarray images were performed using an average of all the gene signals onthe array (global normalization) to normalize the signal intensitybetween arrays. Changes in the mRNA profile of HuB mRNP complexes inresponse to retinoic acid treatment were considered significant if theywere four-fold greater (twice the stringency typically used forestablishing significance of a gene expression change). cDNA arrayimages and overlays were prepared using Adobe Photoshop® 5.0.2 (SanJose, Calif., USA).

EXAMPLE 4 Identification of mRNA Subsets Associated with RNA BindingProteins En Masse Using CDNA Arrays: Experimental Results

Results. After assessing the overall gene expression profile of HuBtransfected P19 cells (the transcriptome), HuB and PABP mRNA complexes,as well as e1F-4E mRNP complexes were separately immunoprecipitated andcaptured mRNAs were identified on cDNA arrays. The initial alignment ofthese arrays was facilitated by spiking the hybridization reaction withradiolabeled lambda phage markers that hybridized with six DNA spots onthe bottom of the array membrane. Once the alignment register wasestablished, subsequent blots did not require the use of spiked lambdamarkers for orientation.

Arrays generated from immunoprecipitations with rabbit pre-bleed serawere essentially blank with the exception of the spiked lambda markersobserved at the bottom of the array (FIG. 5A). Immunoprecipitated HuBmRNP and eIF-4E mRNP complexes each contained slightly more than 10% ofthe mRNAs detected in total cell RNA, but differed considerably from oneanother (FIGS. 5B, 5C, and 5E).

Like HuB and e1F-4E, PABP has been implicated in facilitating mRNAstabilization and translation (Ross (1995) Microbiol. Rev. 59, 423-450;Ross (1996) Trends Genet. 12, 171-175; Wickens et al. (1996) inTranslational control, eds Hershey, J. W. B, Mathews, M. B. & Sonenberg,N., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp.411-450; Sachs et al. (1997) Cell 89, 831-838). Not surprisingly, PABPmRNPs contained many more detectable mRNAs than those observed in theHuB or e1F-4E mRNPs (FIG. 5D). As expected, the profile of the mRNAs inthe PABP mRNPs from these cells closely resembled that of thetranscriptome. However, as was seen for HuB and e1F-4E mRNPs, some mRNAswere enriched or depleted in the PABP-mRNPs as compared to the total RNA(FIGS. 5D and 5E). The profiles and relative abundance of mRNAs detectedin these mRNP complexes were highly reproducible, but the absolutenumber of mRNA species detectable on the phosphorimages occasionallyvaried as a result of differences in the specific activity of the probe.

Because the cDNA arrays derived using total RNA were generated usingone-tenth the quantity of lysate used for mRNP immunoprecipitations, acomparison of the absolute quantities of each mRNA detected in mRNPcomplexes with those observed in the total RNA was not conducted. A moreaccurate result was obtained by comparing the relative abundance of eachmRNA species to each other within each microarray. For example, therelative abundance of the mRNA encoding β-actin and ribosomal proteinS29 (FIG. 5, arrows a and b, respectively) are approximately equal tothe total cellular RNA, but varied dramatically among each of the mRNPcomplexes. Many other examples of this distinction are readily apparentin FIG. 4. These findings indicated that the mRNA profiles detected inHuB, e1F-4E, and PABP mRNP complexes are distinct from one another andfrom those of the transcriptome.

EXAMPLE 5 Alterations in mRNP Complexes in Response to Retinoic Acid(RA)

Since HuB is predominantly a neuronal protein believed to play a role inregulating neuronal differentiation, studies were conducted toinvestigate whether the mRNA population found in HuB mRNP complexeschanges in response to RA, a chemical inducer of neuronaldifferentiation. HuB transfected P19 cells were treated with RA toinduce the onset of neuronal differentiation, HuB mRNP complexes wereimmunoprecipitated, and then associated mRNAs were identified on cDNAarrays. Comparison of the mRNA profiles extracted from the HuB mRNPsbefore and after RA treatment revealed that eighteen mRNAs were eitherexclusively present or greatly enriched (four-fold or greater) inRA-treated HuB mRNPs (FIGS. 6A, 6B, and 6C, red bars). In addition,three mRNAs (T-lymphocyte activated protein, DNA-binding protein SATB1,and HSP84) decreased in abundance by four-fold or greater in response toRA treatment (FIG. 6C, blue bars). To determine if the changes observedin the mRNA profile of the HuB mRNA complexes were unique, theubiquitously expressed ELAV family member HuA (HuR) wasimmunoprecipitated from these RA treated cells. Although there were afew changes to the HuA mRNP profile following treatment with RA, theywere minor in comparison with HuB.

The changes in the HuB-associated mRNA profile in response to RAtreatment did not merely reflect changes in the total cellular mRNA(FIGS. 6G, 6H, and 6I). Numerous examples of differentially-enriched ordepleted mRNAs detected in HuB mRNP complexes are evident by comparingFIGS. 6C and 6I. For comparative purposes, this is depicted in FIG. 7 byrealignment and enlargement of representative spots. For example, IGF-2mRNA was detectable only in total RNA and HuB mRNP complexes fromRA-treated cells (FIG. 7). However, other HuB-mRNP-bound mRNAs, such asintegrin beta, cyclin D2, and Hsp84 increased or decreased in abundancedisproportionately to their changes in the total RNA profile followingRA treatment (FIG. 7). The disparity between changes in the mRNAprofiles of total RNA and HuB mRNPs possibly results from changes incompartmentalization of mRNAs that flux dynamically through mRNPcomplexes in response to RA treatment. It can be concluded that the mRNAprofiles derived from these mRNP complexes are dynamic and can reflectthe state of growth, as well as changes in the cellular environment inresponse to a biological inducer like retinoic acid.

EXAMPLE 6 In vivo Target Sequence Preferences For RNA-Binding Proteins

Using GenBank and EST databases, the 3′ UTR sequences from mRNAsenriched in RA-treated HuB-mRNP complexes were identified (TABLE 2).

TABLE 2 Gene 3′UTR Consensus Sequence CD44 AUUUUCUAUUCCUUU UUUAUUUUAUGUCAUUUUUUUA [SEQ ID NO: 1] IGF-2 UAAAAAACCAAA UUUGAUU GGCUCUAAACA[SEQ ID NO: 2] UAAAGAA AUUAAUU GGCUAAAAACAUA [SEQ ID NO: 3] CUAAAAAUUAAUU GGCUUAAAAA [SEQ ID NO: 4] HOX2.5 UCACUCUU AUUAUUU AU [SEQ ID NO:5] AAAU UUUAUUA AGUUA [SEQ ID NO: 6] AUCAGG UUCAUUU UGGUUGU [SEQ ID NO:7] Inhibitor AU UUUAUCU GUUA [SEQ ID NO: 8] J6 UUUUGUUUUUCUCCCUUUUUUAGUUU UUUCAAA [SEQ ID NO: 9] GADD45 UAUUUUUUUUCUUUUUUUU UUUUGGU CUUUAU[SEQ ID NO: 10] UUAAAUUCUCAGAAGU UUUAUUA UAAAUCUU [SEQ ID NO: 11] Nexin1 UUCUGUUAAAUAUU UUUAUAU ACUGCUUUCUUUUUU [SEQ ID NO: 12] AUUUUAUAGUAGUUUUUAUGU UUUUAUGGAAAA [SEQ ID NO: 13] AUUUGCCUU UUUAAUU CUUUUU [SEQ IDNO: 14] Egr-1 UAUUUUGUGGU UUUAUUU UACUUUGUACUU [SEQ ID NO: 15] Zif268 UUUUGUUU UCCUU [SEQ ID NO: 16] Neuronal- UUU UUUAUUU UCUGUAUUUUUU [SEQ IDNO: 17] Cadherin UUUUUUUUAAAUUUU UUUAUUU UCUUUUU [SEQ ID NO: 18]UUUUUUAUUUUC UGUAUUU UUU [SEQ ID NO: 19] UUUUUAAUUU UUUAAUU UUUUUU [SEQID NO: 20] Integrin alpha AAUGG UUUAUAU UUAUGAU [SEQ ID NO: 21] 5 UUGUUUAUAU CUUCAAU [SEQ ID NO: 22] SEF2 UUCAAGCGC UUGANUU [SEQ ID NO: 23]Cf2r UGCAUCGAUCCG UUGAUUU ACUACU [SEQ ID NO: 24] Integrin UAUAAUUUUUAAUUU UUUAUUAUUUU [SEQ ID NO: 25] Beta UUAUUUUACCUUUUUUUUUUUUC UUUAAUUCCUGGU [SEQ ID NO: 26] CTCF UUAUGAAUGU UAUAUUU GU [SEQ ID NO: 27] UCUUAAUUU UUUCUCUUUUUUUUCUUU [SEQ ID NO: 28] TGF beta 2 UUUUUUUUUCCUUUUAAUU GUAAAUGGUUCUUU [SEQ ID NO: 29] UUAAUGAUCAUUCAGAUUGUA UAUAUUUGUUUCCUUU [SEQ ID NO: 30] UUCAAUUUUU UUUAUAU ACUAUCUU [SEQ ID NO: 31]UUUUUC-- UUUAAUU GGUUUUUUU [SEQ ID NO: 32] MTP UGUCUUGUCUGAGCA UUUAUUUUCAAA [SEQ ID NO: 33] UUCUCGUCUUG UUUAUUU UACAA [SEQ ID NO: 34]UAUAAUAAUAG UUUAUGU UUUGGAUGUUUGGU [SEQ ID NO: 35] Cyclin D2AUGUCUUGUUCUU UGUGUUU UUAGGAU [SEQ ID NO: 36] (AU/GA) UUUAUUU (UA/AG)[SEQ ID NO: 37] In Vitro Consensus Sequence

Many of the mRNAs for which 3′ UTR sequences were available containedsimilar uridylate-rich motifs as those previously found to bind to Huprotein in vitro (Levine et al. (1993) Mol. Cell Biol. 13, 3494-3504;Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11207-11211; King et al(1994) J. Neurosci. 14, 1943-1952; Liu et al. (1995) Neurology 45,544-550; Ma et al. (1997) Nucleic Acids Res. 25, 3564-3569; Fan et al.(1997) Genes Dev. 11, 2557-2568). Moreover, most of these mRNAs encodeproteins that are expressed in neuronal tissues or are known to beup-regulated following RA-induced neuronal differentiation (Beck et al.(1995) Neuron 14, 717-730; Colon and Rossant (1992) Development 116,357-368; Graham et al. (1991) Development 112, 255-264; Hirsch et al.(1994) Dev. Dyn. 201, 108-120; Hunt et al. (1991) Development 112,43-50; Janssen-Timmen et al. (1989) Gene 80, 325-336; Kondo et al.(1992) Nucleic Acids Res. 20, 5729-5735; Konishi et al. (1994) BrainRes. 649, 53-61; Neuman et al. (1993) Eur. J. Neurosci. 5, 311-318;Okuda et al. (1995) Genomics 29, 623-630; Soosaar et al. (1994) BranRes. Mol. Brain Res. 25, 176-180; Takechi et al. (1992) Eur. J. Biochem.206, 323-329; Telford et al. (1990) Mol. Reprod. Dev. 27, 81-92;Zwartkrius et al. (1993) Exp. Cell Res. 205, 422-425; Tomaselli et al.(1988) Neuron 1, 33-43; Redies (1995) Exp. Cell Res. 220, 243-256; Rosset a. (1996) J. Neurosci. 16, 210-219). The sequence alignment shown inTABLE 2 is consistent with the previous results of Levine et al. ((1993)Mol. Cell Biol. 13, 2494-3504) and Gao et al. ((1994) Proc. Natl. Acad.Sci. USA 91, 11207-11211) who used in vitro selection to derive aconsensus RNA-binding sequence for HuB. Using the methods describedherein, it is possible to discern in vivo target sequence preferencesfor other RNA-binding proteins.

EXAMPLE 7 Use Of mRNA Binding Proteins To Purify Endogenous mRNPComplexes and To Identify Associated mRNAs En Masse Using cDNA ArrayAnalysis

Earlier attempts to identify mRNA targets of the HuB protein usinghigh-throughput methods required RT-PCR amplification and in vitroiterative selection and identified several structurally related ERGmRNAs from neuronal tissues (Gao et al. (1994) Proc. Natl. Acad. Sci.USA 91, 11207-11211; Andrews and Keene (1999) Methods Mol. Biol. 118,233-244). Most of these mRNAs contained ARE-like sequences in their3′-UTRs, which is a characteristic of ERG mRNAs (Keene (1999) Proc.Natl. Acad. Sci. USA 96, 5-7; Levine et al. (1993) Moll. Cell Biol. 13,3494-3504; Gao et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11207-11211;King et al. (1994) J. Neurosci. 14, 1943-1952). It has been demonstratedthat Hu proteins can bind ERG mRNAs and affect their stability and/ortranslational activation (Jain et al. (1997) Mol. Cell Biol. 17,954-962; Levy et al. (1998) J. Biol. Chem. 273, 6417-6423; Fan andSteitz (1998) EMBO J. 17, 3448-3460; Peng et al. (1998) EMBO J. 17,3461-3470; Keene (1999) Proc. Natl. Acad. Sci. USA 96, 5-7; Levine etal. (1993) Mol. Cell Biol. 13, 3494-3504; Gao et al. (1994) Proc. Natl.Acad. Sci. USA 91, 11207-11211; King et al. (1994) J. Neurosci. 14,1943-1952; Liu et al. (1995) Neurology 45, 544-550; Chung et al. (1997)J. Biol. Chem. 272, 6593-6598; Antic et al. (1999) Genes Dev. 13,449-461; Ma et al. (1997) Nucleic Acids Res. 25, 3564-3569; Aranda-Abreuet al. (1999) J. Neurosci. 19, 6907-6917). The in vitro approach of Gaoet al. ((1994) Proc. Natl. Acad. Sci. USA 91, 11207-11211) yielded adistinct mRNA subset from human brain and medulloblastoma cells with ERGsequence characteristics. This more direct in vivo approach obviates theneed for in vitro binding and PCR amplification. Moreover, this newapproach allows the identification of mRNA transcripts with linkedstructural and functional properties, may of which would not be detected(and could not be detected) using in vitro techniques. In addition,recognizable HuB protein-RNA binding sequences were identified withinthe in vivo-captured mRNA subset (TABLE 2).

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

1. A method of characterizing an RNA-protein complex, the methodcomprising: a) expressing in vivo an epitope-tagged RNA-binding proteinthereby forming an RNA-protein complex in a cell, b) lysing the cell toproduce a lysate; c) contacting the lysate with a ligand thatspecifically binds to the epitope tag; d) isolating the RNA-proteincomplex from the lysate; and e) identifying at least one RNA and/or atleast one protein, other than the epitope-tagged protein, from theisolated the RNA-protein complex; wherein the nucleic acid encoding theepitope-tagged RNA-binding protein is under the control of atissue-specific promoter.